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RNAi and SiRNA Forum Discuss and Post Questions in this forum about RNA interference and siRNA. RNAi and siRNA transfection, inhibition of endogenous genes, and miRNA and shRNA.


gene silencing- protein product

gene silencing- protein product - RNAi and SiRNA Forum

gene silencing- protein product - Discuss and Post Questions in this forum about RNA interference and siRNA. RNAi and siRNA transfection, inhibition of endogenous genes, and miRNA and shRNA.


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  #1  
Old 07-15-2014, 09:06 AM
Pipette Filler
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Default gene silencing- protein product



hello!!

I have succeed silencing a gene with siRNA and confirmed it in Real Time PCR.

the thing is that in Western blot the protein encodes from that gene still expressed...

how could it be?!

thank you.
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Old 07-21-2014, 03:28 PM
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Default Re: gene silencing- protein product

It can take some time for the pre-existing protein to degrade. When you halt gene expression, the amount of remaining protein should drop as the protein is degraded, but the rate of degradation varies between proteins. Do you know the expected half-life of the protein? How long did you wait between the knockdown and harvesting the protein?
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Old 07-22-2014, 07:52 AM
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Default Re: gene silencing- protein product

the protein I'm working on is STAT3 wich is a transcription factor with a short half life (don't know the exact time).
between the transfection and the harvesting there is 48h/72h...
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Old 07-22-2014, 03:43 PM
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Default Re: gene silencing- protein product

A delay of 2 to 3 days between initiating an knockdown and harvesting a protein should be plenty of time to see a decrease in a transcription factor by Western blot. I don't think I can help you further on this, as my area of work is antisense and not siRNA; your problem sounds specific to the siRNA technique.

Is there an siRNA person who can jump in and help?
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Old 09-03-2014, 06:15 AM
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Default Re: gene silencing- protein product

Are there any isoforms of your gene? It could be that the siRNA is knocking down one of them (the one you are targeting with your siRNA), while your antibodies recognize other isoforms that do not have the siRNA target region in their sequence.
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